C-terminally truncated interferon alpha variants

ABSTRACT

The present invention provides biologically active variants of human α-2b-interferon. The variants contain carboxy terminus truncations when compared with the amino acid sequence of full-length human α-2b-interferon. It is the novel finding of the present invention that these truncated variants have the biological activity of full-length human α-2b-interferon. The invention encompasses these biologically active variant α-interferons, as well as polynucleotides encoding these interferons. Expression cassettes comprising these polynucleotides and host cells comprising the expression cassettes are also provided. The invention also provides compositions comprising variant α-interferon polypeptides and a pharmaceutically acceptable carrier.

FIELD OF THE INVENTION

The present invention relates to biologically active variants of humanalpha interferon.

BACKGROUND OF THE INVENTION

The interferons are a family of glycoproteins whose secretion from cellsis induced by a number of signals including viruses, double-strandedRNAs, other polynucleotides, antigens, and mitogens. Interferons exhibitmultiple biological activities, including antiviral, antiproliferative,and immunomodulatory activities. At least three distinct types of humaninterferons, α, β, and γ, have been distinguished based on a number offactors, including anti-viral and anti-proliferative activities.

α-interferons act through interaction with cell-surface receptors andinduce the expression, primarily at the transcriptional level, of abroad but specific set of cellular genes. Several INTERFERON-inducedgene products have been used as markers for the biological activity ofinterferons. These include, for instance, ISG15, ISG54, IRF1, GBP, andIP10.

Assays for interferon-mediated anti-viral activity have been describedin the art. See, for example, McNeill, (1981) J Immunol Methods.46:121-7. Assays for interferon antiviral activity include inhibition ofcytopathic effect, virus plaque formation; and reduction of virus yield.Viral cytopathic effect assays measure the degree of protection inducedin cell cultures pretreated with interferon INTERFERON and subsequentlyinfected with viruses. See, for example, Rubinstein et al. (1981) JVirol. 37:755-8. Plaque-reduction assays can be used to measure theresistance of INTERFERON-treated cell cultures to a plaque-forming virus(for instance, measles virus). Finally, virus yield assays measure theamount of virus released from cells during, for instance, a singlegrowth cycle. Such assays are useful for testing the antiviral activityof INTERFERONs against viruses that do not cause cytopathic effects, orthat do not build plaques in target-cell cultures.

α-2b interferons have since been shown to be efficacious against viral,proliferative, and inflammatory disorders, including malignant melanoma,hairy cell leukemia, chronic hepatitis B, chronic hepatitis C,condylomata acuminata, follicular (non-Hodgkin's) lymphoma, andAIDS-related Kaposi's sarcoma. Clinical uses of interferons are reviewedin Gresser (1997) J. Leukoc. Biol. 61:567-74, and Pfeffer (1997) SeminOncol. 24(3 Suppl 9):S9-63-S9-69.

SUMMARY OF THE INVENTION

The present invention provides biologically active variants of humanα-2b-interferon. The variants contain carboxy terminus truncations whencompared with the amino acid sequence of full-length humanα-2b-interferon. It is the novel finding of the present invention thatthese truncated variants have the biological activity of humanα-2b-interferon. The sequences of the α-2b-interferon variant precursorpolypeptides are shown in SEQ ID NOS:1-5, while the sequences of themature α-2b-interferon variant polypeptides s are shown in SEQ IDNOS:6-10. Accordingly, in one embodiment, the invention provides apurified polypeptide consisting of an amino acid sequence selected fromthe amino acid sequences shown in SEQ ID NOS:1-10.

In some embodiments, the polypeptide consisting of a signal peptideoperably linked to an amino acid sequence selected from the sequencesshown in SEQ ID NOS:6-10. In some embodiments, the signal peptide is amammalian signal peptide, while in other embodiments, the signal peptideis a plant signal peptide.

In one aspect of the invention, the polypeptides of the invention arerecombinantly produced in a host cell plant cell. In particularembodiments, the host cell is a mammalian cell, a plant cell, a yeastcell, an insect cell, or a prokaryotic cell.

The invention also encompasses polynucleotides encoding the polypeptideof the invention, expression cassettes comprising these polynucleotides,and host cells comprising the expression cassettes.

In another embodiment, the invention provides a composition comprising apurified polypeptide of the invention and a pharmaceutically acceptablecarrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 show the interferon levels (as determined by a solid phasesandwich immunoassay) in the media and tissue of a transformed duckweedculture, as described Example 1.

FIG. 2 show the interferon levels (as determined by a solid phasesandwich immunoassay) in the media and tissue of a transformed duckweedcultures, as described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides biologically active variants of humanα-2b-interferon. The variants contain carboxy terminus truncations whencompared with the amino acid sequence of full-length humanα-2b-interferon. It is the novel finding of the present invention thatthese truncated variants have the biological activity of humanα-2b-interferon. The present invention provides the sequences of theseα-2b interferon variants.

DEFINITIONS

An “isolated” or “purified” polynucleotide or polypeptide issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or polypeptide is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” polynucleotide is freeof sequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-polypeptide-of-interest chemicals.

A “biologically active polypeptide” refers to a polypeptide that has thecapability of performing one or more biological functions or a set ofactivities normally attributed to the polypeptide in a biologicalcontext. Those skilled in the art will appreciate that the term“biologically active” includes polypeptides in which the biologicalactivity is altered as compared with the native protein (e.g.,suppressed or enhanced), as long as the protein has sufficient activityto be of interest for use in industrial or chemical processes or as atherapeutic, vaccine, or diagnostics reagent. Biological activity can bedetermined by any method available in the art. For example, thebiological activity of members of the interferon family of proteins canbe determined by any of a number of methods including their interactionwith interferon-specific antibodies, their ability to increaseresistance to viral infection, or their ability to modulate thetranscription of interferon-regulated gene targets. Examples of suchmethods are described elsewhere herein.

The terms “expression” or “production” refer to the biosynthesis of agene product, including the transcription, translation, and assembly ofsaid gene product.

By “recombinantly produced” is intended a polypeptide that has beenprepared by recombinant DNA techniques. Recombinantly producedinterferon variants can be produced by culturing a host cell transformedwith an expression cassette comprising a polynucleotide that encodes anα-interferon variant of the invention. The host cell is one that cantranscribe the nucleotide sequence and produce the desired protein, andcan be prokaryotic (for example, E. coli) or eukaryotic (for example aplant, yeast, insect, or mammalian cell).

The term “duckweed” refers to members of the family Lemnaceae. Thisfamily currently is divided into five genera and 38 species of duckweedas follows: genus Lemna (L. aequinoctialis, L. disperma, L.ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L.obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L.valdiviana); genus Spirodela (S. intermedia, S. polyrrhiza); genusWolfia (Wa. angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa.brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa, Wa.microscopica, Wa. neglecta) genus Wolfiella (Wl. caudata, Wl.denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata, Wl. repunda, Wl.rotunda, and Wl. neotropica), and genus Landoltia (L. punctata). Anyother genera or species of Lemnaceae, if they exist, are also aspects ofthe present invention. Lemna species can be classified using thetaxonomic scheme described by Les et al. (2002) Systematic Botany27:221-40.

“Operably linked” as used herein in reference to nucleotide sequencesrefers to multiple nucleotide sequences that are placed in a functionalrelationship with each other. Generally, operably linked DNA sequencesare contiguous and, where necessary to join two protein coding regions,in reading frame.

A. Polypeptides.

The present invention identifies biologically active variants of humanα-2b interferon. These variants contain carboxy terminus truncations of4-8 amino acids in comparison with full-length human α-2b-interferon.The sequences of the mature forms of these biologically activeinterferon variants are provided in SEQ ID NOS:6-10, while thecorresponding sequences of the interferon variant precursor polypeptidesare provided in SEQ ID NOS:1-5.

In some embodiments, the invention encompasses compositions comprising amixture of two or more α-interferon variants of the invention. Suchmixtures may comprise two or more, three or more, four or more, five ormore, or more than six of the α-interferon variants consisting of theamino acid sequences set forth in SEQ ID NOS:1-10.

B. Polynucleotides and Expression Cassettes

In one aspect the present invention provides polynucleotides encodingthe biologically active α-interferons of the invention. Accordingly, theinvention encompasses polynucleotides encoding polypeptides consistingof the amino acid sequences set forth in SEQ ID NOS:1-10.

In some embodiments, the polynucleotides may be comprised within anexpression cassette. The expression cassette comprises a transcriptionalinitiation region linked to the nucleic acid or gene of interest. Suchan expression cassette can be provided with a plurality of restrictionsites for insertion of the polynucleotide of interest to be under thetranscriptional regulation of the regulatory regions.

The transcriptional initiation region, (e.g., a promoter) may be nativeor homologous or foreign or heterologous to the host, or could be thenatural sequence or a synthetic sequence. By foreign, it is intendedthat the transcriptional initiation region is not found in the wild-typehost into which the transcriptional initiation region is introduced. Asused herein a chimeric gene comprises a coding sequence operably linkedto a transcription initiation region that is heterologous to the codingsequence.

Any suitable promoter known in the art can be employed according to thepresent invention (including bacterial, yeast, fungal, insect,mammalian, and plant promoters). For example, plant promoters may beused. Exemplary promoters include, but are not limited to, theCauliflower Mosaic Virus 35S promoter, the opine synthetase promoters(e.g., nos, mas, ocs, etc.), the ubiquitin promoter, the actin promoter,the ribulose bisphosphate (RubP) carboxylase small subunit promoter, andthe alcohol dehydrogenase promoter. The duckweed RubP carboxylase smallsubunit promoter is known in the art (Silverthorne et al. (1990) PlantMol. Biol. 15:49). Other promoters from viruses that infect plants,preferably duckweed, are also suitable including, but not limited to,promoters isolated from Dasheen mosaic virus, Chlorella virus (e.g., theChlorella virus adenine methyltransferase promoter; Mitra et al. (1994)Plant Mol. Biol. 26:85), tomato spotted wilt virus, tobacco rattlevirus, tobacco necrosis virus, tobacco ring spot virus, tomato ring spotvirus, cucumber mosaic virus, peanut stump virus, alfalfa mosaic virus,sugarcane baciliform badnavirus and the like.

The overall strength of a given promoter can be influenced by thecombination and spatial organization of cis-acting nucleotide sequencessuch as upstream activating sequences. For example, activatingnucleotide sequences derived from the Agrobacterium tumefaciens octopinesynthase gene can enhance transcription from the Agrobacteriumtumefaciens mannopine synthase promoter (see U.S. Pat. No. 5,955,646 toGelvin et al.). In the present invention, the expression cassette cancontain activating nucleotide sequences inserted upstream of thepromoter sequence to enhance the expression of the nucleotide sequenceof interest. In one embodiment, the expression cassette includes threeupstream activating sequences derived from the Agrobacterium tumefaciensoctopine synthase gene operably linked to a promoter derived from anAgrobacterium tumefaciens mannopine synthase gene (see U.S. Pat. No.5,955,646, herein incorporated by reference).

The expression cassette may include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, apolynucleotide of interest, and a transcriptional and translationaltermination region functional in plants. Any suitable terminationsequence known in the art may be used in accordance with the presentinvention. The termination region may be native with the transcriptionalinitiation region, may be native with the nucleotide sequence ofinterest, or may be derived from another source. Convenient terminationregions are available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthetase and nopaline synthetase termination regions. Seealso Guerineau et al. (1991) Mol. Gen. Genet. 262:141; Proudfoot (1991)Cell 64:671; Sanfacon et al. (1991) Genes Dev. 5:141; Mogen et al.(1990) Plant Cell 2:1261; Munroe et al. (1990) Gene 91:151; Ballas etal. (1989) Nucleic Acids Res. 17:7891; and Joshi et al. (1987) NucleicAcids Res. 15:9627. Additional exemplary termination sequences are thepea RubP carboxylase small subunit termination sequence and theCauliflower Mosaic Virus 35S termination sequence. Other suitabletermination sequences will be apparent to those skilled in the art.

Alternatively, the polynucleotides of interest can be provided on anyother suitable expression cassette known in the art.

The expression cassettes may contain more than one polynucleotide to betransferred and expressed in the transformed plant. Thus, each nucleicacid sequence will be operably linked to 5′ and 3′ regulatory sequences.Alternatively, multiple expression cassettes may be provided.

The expression cassette may comprise a selectable marker gene for theselection of transformed cells or tissues. Selectable marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds.Herbicide resistance genes generally code for a modified target proteininsensitive to the herbicide or for an enzyme that degrades ordetoxifies the herbicide in the plant before it can act. See DeBlock etal. (1987) EMBO J. 6:2513; DeBlock et al. (1989) Plant Physiol. 91:691;Fromm et al. (1990) BioTechnology 8:833; Gordon-Kamm et al. (1990) PlantCell 2:603; and Frisch et al. (1995) Plant Mol. Biol. 27:405-9. Forexample, resistance to glyphosphate or sulfonylurea herbicides has beenobtained using genes coding for the mutant target enzymes,5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactatesynthase (ALS). Resistance to glufosinate ammonium, boromoxynil, and2,4-dichlorophenoxyacetate (2,4-D) have been obtained by using bacterialgenes encoding phosphinothricin acetyltransferase, a nitrilase, or a2,4-dichlorophenoxyacetate monooxygenase, which detoxify the respectiveherbicides.

For purposes of the present invention, selectable marker genes include,but are not limited to, genes encoding neomycin phosphotransferase II(Fraley et al. (1986) CRC Critical Reviews in Plant Science 4:1);neomycin phosphotransferase III (Frisch et al. (1995) Plant Mol. Biol.27:405-9); cyanamide hydratase (Maier-Greiner et al. (1991) Proc. Natl.Acad. Sci. USA 88:4250); aspartate kinase; dihydrodipicolinate synthase(Perl et al. (1993) BioTechnology 11:715); bar gene (Toki et al. (1992)Plant Physiol. 100:1503; Meagher et al. (1996) Crop Sci. 36:1367);tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol. Biol.22:907); neomycin phosphotransferase (NEO; Southern et al. (1982) J.Mol. Appl. Gen. 1:327); hygromycin phosphotransferase (HPT or HYG;Shimizu et al. (1986) Mol. Cell. Biol. 6:1074); dihydrofolate reductase(DHFR; Kwok et al. (1986) Proc. Natl. Acad. Sci. USA 83:4552);phosphinothricin acetyltransferase (DeBlock et al. (1987) EMBO J.6:2513); 2,2-dichloropropionic acid dehalogenase (Buchanan-Wollatron etal. (1989) J. Cell. Biochem. 13D:330); acetohydroxyacid synthase (U.S.Pat. No. 4,761,373 to Anderson et al.; Haughn et al. (1988) Mol. Gen.Genet. 221:266); 5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comaiet al. (1985) Nature 317:741); haloarylnitrilase (WO 87/04181 to Stalkeret al.); acetyl-coenzyme A carboxylase (Parker et al. (1990) PlantPhysiol. 92:1220); dihydropteroate synthase (sulI; Guerineau et al.(1990) Plant Mol. Biol. 15:127); and 32 kDa photosystem II polypeptide(psbA; Hirschberg et al. (1983) Science 222:1346 (1983).

Also included are genes encoding resistance to: gentamycin (e.g., aacC1,Wohlleben et al. (1989) Mol. Gen. Genet. 217:202-208); chloramphenicol(Herrera-Estrella et al. (1983) EMBO J. 2:987); methotrexate(Herrera-Estrella et al. (1983) Nature 303:209; Meijer et al. (1991)Plant Mol. Biol. 16:807); hygromycin (Waldron et al. (1985) Plant Mol.Biol. 5:103; Zhijian et al. (1995) Plant Science 108:219; Meijer et al.(1991) Plant Mol. Bio. 16:807); streptomycin (Jones et al. (1987) Mol.Gen. Genet. 210:86); spectinomycin (Bretagne-Sagnard et al. (1996)Transgenic Res. 5:131); bleomycin (Hille et al. (1986) Plant Mol. Biol.7:171); sulfonamide (Guerineau et al. (1990) Plant Mol. Bio. 15:127);bromoxynil (Stalker et al. (1988) Science 242:419); 2,4-D (Streber etal. (1989) BioTechnology 7:811); phosphinothricin (DeBlock et al. (1987)EMBO J. 6:2513); spectinomycin (Bretagne-Sagnard and Chupeau, TransgenicResearch 5:131).

The bar gene confers herbicide resistance to glufosinate-typeherbicides, such as phosphinothricin (PPT) or bialaphos, and the like.As noted above, other selectable markers that could be used in thevector constructs include, but are not limited to, the pat gene, alsofor bialaphos and phosphinothricin resistance, the ALS gene forimidazolinone resistance, the HPH or HYG gene for hygromycin resistance,the EPSP synthase gene for glyphosate resistance, the Hm1 gene forresistance to the Hc-toxin, and other selective agents used routinelyand known to one of ordinary skill in the art. See Yarranton (1992)Curr. Opin. Biotech. 3:506; Chistopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314; Yao et al. (1992) Cell 71:63; Reznikoff (1992)Mol. Microbiol. 6:2419; Barkley et al. (1980) The Operon 177-220; Hu etal. (1987) Cell 48:555; Brown et al. (1987) Cell 49:603; Figge et al.(1988) Cell 52:713; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA86:5400; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549;Deuschle et al. (1990) Science 248:480; Labow et al. (1990) Mol. Cell.Biol. 10:3343; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072; Wyborskiet al. (1991) Nuc. Acids Res. 19:4647; Hillenand-Wissman (1989) Topicsin Mol. And Struc. Biol. 10:143; Degenkolb et al. (1991) Antimicrob.Agents Chemother. 35:1591; Kleinschnidt et al. (1988) Biochemistry27:1094; Gatz et al. (1992) Plant J. 2:397; Gossen et al. (1992) Proc.Natl. Acad. Sci. USA 89:5547; Oliva et al. (1992) Antimicrob. AgentsChemother. 36:913; Hlavka et al. (1985) Handbook of ExperimentalPharmacology 78; and Gill et al. (1988) Nature 334:721. Such disclosuresare herein incorporated by reference.

The above list of selectable marker genes are not meant to be limiting.Any lethal or non-lethal selectable marker gene can be used in thepresent invention.

C. Modification of Nucleotide Sequences for Enhanced Expression in aHost Cell

The present invention provides for the modification of thepolynucleotide to enhance its recombinant production in a host cell. Onesuch modification is the synthesis of the nucleotide sequence ofinterest using codons preferred in the host cell. Methods are availablein the art for synthesizing nucleotide sequences with host-preferredcodons. See, e.g., U.S. Pat. Nos. 5,380,831 and 5,436,391; Perlak et al.(1991) Proc. Natl. Acad. Sci. USA 15:3324; Iannacome et al. (1997) PlantMol. Biol. 34:485; and Murray et al., (1989) Nucleic Acids. Res. 17:477,herein incorporated by reference. The preferred codons may be determinedfrom the codons of highest frequency in the proteins expressed in thehost cell. All or any part of the polynucleotide may be optimized orsynthetic. In other words, fully optimized or partially optimizedsequences may also be used. For example, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% of the codons may be host cell-preferred codons. In oneembodiment, between 90 and 96% of the codons are host cell-preferredcodons.

Other modifications can also be made to the nucleotide sequence ofinterest to enhance its expression in a host cell. These modificationsinclude, but are not limited to, elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well characterized sequenceswhich may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence may be modified to avoid predicted hairpinsecondary mRNA structures.

Expression of a transgene in a host cell can also be enhanced by the useof 5′ leader sequences. Such leader sequences can act to enhancetranslation. Translation leaders are known in the art and include, butare not limited to, picornavirus leaders, e.g., EMCV leader(Encephalomyocarditis 5′ noncoding region; Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci USA 86:6126); potyvirus leaders, e.g., TEV leader(Tobacco Etch Virus; Allison et al. (1986) Virology 154:9); humanimmunoglobulin heavy-chain binding protein (BiP; Macajak and Sarnow(1991) Nature 353:90); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4; Jobling and Gehrke (1987) Nature325:622); tobacco mosaic virus leader (TMV; Gallie (1989) MolecularBiology of RNA, 23:56); potato etch virus leader (Tomashevskaya et al.(1993) J. Gen. Virol. 74:2717-2724); Fed-1 5′ untranslated region(Dickey (1992) EMBO J. 11:2311-2317); RbcS 5′ untranslated region(Silverthorne et al. (1990) J. Plant. Mol. Biol. 15:49-58); and maizechlorotic mottle virus leader (MCMV; Lommel et al. (1991) Virology81:382). See also, Della-Cioppa et al. (1987) Plant Physiology 84:965.Leader sequence comprising plant intron sequence, including intronsequence from the maize dehydrogenase 1 gene, the castor bean catalasegene, or the Arabidopsis tryptophan pathway gene PAT1 has also beenshown to increase translational efficient in plants (Callis et al.(1987) Genes Dev. 1:1183-1200; Mascarenhas et al. (1990) Plant Mol.Biol. 15:913-920). Other leader sequences that may be used include theleader from the Lemna gibba ribulose-bis-phosphate carboxylase smallsubunit 5B gene (Buzby et al. (1990) Plant Cell 2:805-814).

D. Signal Peptides

Secreted proteins including interferon are usually translated fromprecursor polypeptides that include a “signal peptide” that interactswith a receptor protein on the membrane of the endoplasmic reticulum todirect the translocation of the growing polypeptide chain across themembrane and into the endoplasmic reticulum for secretion from the cell.This signal peptide is often cleaved from the precursor polypeptide toproduce a “mature” polypeptide lacking the signal peptide. In anembodiment of the present invention, a biologically active interferonvariant is expressed in duckweed from a polynucleotide that is operablylinked with a nucleotide sequence encoding a signal peptide that directssecretion of the interferon variant from the host cell. Any signalpeptide known in the art can be used according to the present invention.Plant signal peptides that target protein translocation to theendoplasmic reticulum (for secretion into the apoplast or outside of thecell) are known in the art. See, for example, U.S. Pat. No. 6,020,169 toLee et al. Alternatively, a mammalian signal peptide can be used totarget recombinant interferon variants expressed in a host cell forsecretion. In one embodiment of the present invention, the mammaliansignal peptide that targets polypeptide secretion is the humanα-2b-interferon signal peptide (amino acids 1-23 of NCBI ProteinAccession No. AAB59402 and SEQ ID NO:12).

In one embodiment, the nucleotide sequence encoding the signal peptideis modified for enhanced expression in the host cell, utilizing anymodification or combination of modifications disclosed in section Cabove for the polynucleotides of interest.

The secreted biologically active polypeptide can be harvested from thehost cell or host cell culture medium by any conventional means known inthe art and purified by chromatography, electrophoresis, dialysis,solvent-solvent extraction, and the like.

E. Host cells

In some embodiments, the invention encompasses host cells containing theexpression cassettes of the invention. These host cells may be used torecombinantly produce the α-interferon variants. The host cell is onethat can transcribe the polynucleotide and can be prokaryotic (forexample, E. coli) or eukaryotic (for example a plant, yeast, insect, ormammalian cell). Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Methods of transforming such host cells with anucleic acid molecule are well known in the art.

In some embodiments, the host cells are plant cells. Both monocot cellsand dicot cells may be used. Suitable methods of introducingpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al.,U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984)EMBO J. 3:2717-2722), and ballistic particle acceleration (see, forexample, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S.Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney etal., U.S. Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transferinto Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926); and Lec1 transformation (WO 00/28058).

In particular embodiments, the host cells for recombinant expression ofthe α-interferon variants are duckweed cells. Stably transformedduckweed cells may also be obtained according any method known in theart. See, for example, U.S. Pat. No. 6,040,498, and PCT publicationsWO0210414 and WO02097433.

When the host cell is a plant cell, transgenic plants can be regeneratedfrom transformed host cells.

F. Pharmaceutical Compositions

The α-interferon variants of the invention can be incorporated intopharmaceutical compositions. Such compositions typically include one ormore α-interferon variant polypeptides and a pharmaceutically acceptablecarrier. The phrase “pharmaceutically acceptable carrier” as used hereinis a carrier that is conventionally used in the art to facilitate thestorage, administration, and/or the healing effect of the therapeuticingredients. A carrier may also reduce any undesirable side effects ofthe α-interferon.

A carrier should be stable (i.e., incapable of reacting with otheringredients in the composition), and it should not produce adverseeffects in patients at the dosages and concentrations employed fortreatment. Suitable carriers include large stable macromolecules such asalbumin, gelatin, collagen, polysaccharide, monosaccharides,polyvinyl-pyrrolidone, polylactic acid, polyglycolic acid, polymericamino acids, fixed oils, ethyl oleate, liposomes, glucose, sucrose,lactose, mannose, dextrose, dextran, cellulose, sorbitol, polyethyleneglycol (PEG), and the like. Slow-release carriers, such as hyaluronicacid, may also be suitable.

Other acceptable components in the composition include, but are notlimited to, pharmaceutically acceptable agents that modify isotonicityincluding water, salts, sugars, polyols, amino acids, and buffers.Examples of suitable buffers include phosphate, citrate, succinate,acetate, and other organic acids or their salts and salts that modifythe tonicity such as sodium chloride, sodium phosphate, sodium sulfate,potassium chloride, and can also include the buffers listed above.

The pharmaceutical composition may additionally comprise a solubilizingagent or solubility enhancer. Examples of such solubility enhancers aredescribed, for example, in Wang et al. (1980) J. Parenteral Drug Assoc.34:452-462; herein incorporated by reference.

Non-limiting examples of solubilizing agents encompassed by the presentinvention include surfactants (detergents) that have a suitablehydrophobic-hydrophilic balance to solubilize interferon. Strong naturalor synthetic anionic surfactants such as alkali metal salts of fattyacids and alkali metal alkyl sulfates may be used. Examples of othersolubilizing agents that can be used in compositions of the inventioninclude but are not limited to sodium dodecyl sulfonate, sodium decylsulfate, sodium tetradecyl sulfate, sodium tridecyl sulfonate, sodiummyristate, sodium caproylate, sodium dodecyl N-sarcosinate, and sodiumtetradecyl N-sarcosinate. Classic stabilization of pharmaceuticals bysurfactants or emulsifiers is described, for example, in Levine et al.(1991) J. Parenteral Sci. Technol. 45(3):160-165. Additional suitablesurfactants are discussed in U.S. Pat. No. 5,935,566, hereinincorporated by reference.

In addition to those agents disclosed above, other stabilizing agents,such as ethylenediaminetetracetic acid (EDTA) or one of its salts suchas disodium EDTA, can be added to further enhance the stability of thepharmaceutical compositions. The EDTA acts as a scavenger of metal ionsknown to catalyze many oxidation reactions, thus providing an additionalstabilizing agent.

Where the composition is used for delivery to a mammal such as a human,the isotonicity of the composition is also a consideration. Thus, in oneembodiment, the composition for an injectable solution will provide anisotonicity the same as, or similar to, that of patient serum or bodyfluids. To achieve isotonicity, a salt, such as sodium chloride,potassium, chloride, or a phosphate buffer, can be added to the solutionat an appropriate concentration.

The pH of the composition is also a consideration. The compositions ofthe invention have a pH ranging from about 4.0 to about 8.5. Suitable pHranges include, for example, about 4.5 to about 7.8 or about 5.0 toabout 7.5 such as about 6.0, about 6.2, about 6.4, about 6.6, about 6.7,about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about7.4, about 7.5, or about 7.6.

A thorough discussion of formulation and selection of pharmaceuticallyacceptable carriers, stabilizers, etc. can be found in Remington'sPharmaceutical Sciences (1990) (18th ed., Mack Pub. Co., Eaton, Pa.),herein incorporated by reference.

EXPERIMENTAL

The following examples are offered for purposes of illustration, not byway of limitation.

Expression Vectors

The expression vector pBMSP-1 used in some of the examples is describedin U.S. Pat. No. 5,955,646, herein incorporated by reference. ThepBMSP-1 transcriptional cassette contains three copies of atranscriptional activating nucleotide sequence derived from theAgrobacterium tumefaciens octopine synthase and, an additionaltranscriptional activating nucleotide sequence derived from theAgrobacterium tumefaciens mannopine synthase gene, a promoter regionderived from the Agrobacterium tumefaciens mannopine synthase gene, apolylinker site for insertion of the nucleotide sequence encoding thepolypeptide of interest, and a termination sequence derived from theAgrobacterium tumefaciens nopaline synthase gene (see, van Engelen etal. (1995) 4:288-290; Ni et al. (1995) Plant J. 7:661-76; and Luehrsenet al. (1991) Mol. Gen. Genet. 225:81-93, each of which is hereinincorporated by reference). The pBMSP-1 expression vector also containsa nucleotide sequence coding for neomycin phosphotransferase II as aselectable marker. Transcription of the selectable marker sequence isdriven by a promoter derived from the Agrobacterium tumefaciens nopalinesynthase gene.

The expression vector pBMSP-3, also used in some of the followingexamples, contains the components of the pBMSP-1 expression vectordescribed above and additionally contains a nucleotide sequencecorresponding to nucleotides 1222-1775 of the maize alcoholdehydrogenase gene (GenBank Accession Number X04049) inserted betweenthe promoter and the polylinker.

Expression Constructs for the Production of Human α-2b-Interferon inDuckweed

Table 2 shows the expression constructs used for the production of humanα-interferon in duckweed.

TABLE 2 Construct Expression Interferon-encoding Name Vector SignalPeptide Sequence IFN01 pBMSP-1 None Non-optimized human α- 2b-interferonIFN02 pBMSP-3 Non-optimized human α-2b- Non-optimized human α-interferon signal peptide 2b-interferon IFN03 pBMSP-3 Arabidopsisthaliana Non-optimized human α- endochitinase signal peptide2b-interferon (nucleotides 338-399 of GenBank Accession number AB023460with an additional “A” added to the 3′ end of the sequence) IFN05pBMSP-3 Modified rice α-amylase signal Non-optimized human α- peptide*2b-interferon IFN07 pBMSP-3 Wild type rice α-amylase signalNon-optimized human α- peptide (nucleotides 34-126 of 2b-interferonGenBank Accession No. M24286) IFN08 pBMSP-3 Duckweed codon optimizedwild Non-optimized human α- type rice α-amylase signal 2b-interferonpeptide IFN09 pBMSP-3 Duckweed codon optimized wild Duckweed codonoptimized type rice α-amylase signal human α-2b-interferon peptide IFN10pBMSP-3 None Duckweed codon optimized human α-2b-interferon IFN11pBMSP-1 Duckweed codon optimized wild Duckweed codon optimized type riceα-amylase signal human α-2b-interferon peptide IFN12 pBMSP-1 NoneDuckweed codon optimized human α-2b-interferon IFN053 modified Duckweedcodon optimized wild Duckweed codon optimized pBMSP-3** type riceα-amylase signal human α-2b-interferon peptide *The nucleotide sequenceencoding the modified rice α-amylase signal peptide corresponds tonucleotides 34-126 of NCBI Accession No. M24286, except that nucleotides97-102 have been changed from “CTTGGC” to “ATCGTC.” **For constructIFN053, the 5′-mas leader in pBMPSP3 was replaced with the leader fromthe ribulose-bis-phosphate carboxylase small subunit 5B gene of Lemnagibba (nucleotides 689-751 of NCBI Accession No. S45167, Buzby et al.(1990) Plant Cell 2: 805-814).Transformation of Duckweed

Duckweed fronds or duckweed nodule cultures (derived from Lemna minorstrain 8627 in these examples) were transformed with the expressionconstructs described above using Agrobacteria-mediated transformationmethods. Agrobacterium tumefaciens strain C58Z707, a disarmed, broadhost range C58 strain (Hepburn et al. (1985) J. Gen. Microbiol.131:2961-2969) is used for transformation in these examples. Theexpression constructs described above were mobilized into A. tumefaciensby electroporation, or by a triparental mating procedure using E. coliMM294 harboring the mobilizing plasmid pRK2013 (Hoekema et al. (1983)Nature 303: 179-180; Ditta et al. (1980) Proc. Natl. Acad. Sci. USA 77:7347-7350). C58Z707 strains comprising the expression constructsdescribed above are streaked on AB minimal medium (Chilton et al.,(1974) Proc. Nat. Acad. Sci. USA 71: 3672-3676) or in YEB medium (1 g/Lyeast extract, 5 g/L beef extract, 5 g/L peptone, 5 g/L sucrose, 0.5 g/LMgSO₄) containing streptomycin at 500 mg/L, spectinomycin at 50 mg/L andkanamycin sulfate at 50 mg/L and grown overnight at 28° C.

In these examples, Lemna minor strain 8627 was used for transformationalthough any Lemna strain can be used. Fronds were grown on liquidSchenk and Hildebrandt medium (Schenk and Hildebrandt (1972) Can. J.Bot. 50:199) containing 1% sucrose and 10 μM indoleacetic acid at 23° C.under a 16-hour light/8-hour dark photoperiod with light intensity ofapproximately 40 μM/m²·sec. For inoculation, individual fronds wereseparated from clumps and floated in inoculation media for approximately2 to 20 minutes. The inoculating medium is Schenk and Hildebrandt medium(pH 5.6) supplemented with 0.6 M mannitol and 100 μM acetosyringone,with the appropriate Agrobacterium tumefaciens strain comprising theexpression construct present at a concentration of about 1×10⁹ cells/ml.These fronds were then transferred to Schenk and Hildebrandt medium (pH5.6) containing 1% sucrose, 0.9% agar, and 20 mg/L acetosyringone andare co-cultivated for 3 or 4 days in the dark at 23° C.

Following co-cultivation, the fronds were transferred for recovery toSchenk and Hildebrandt medium or Murashige and Skoog medium (Murashigeand Skoog (1962) Physiol. Plant. 15:473) supplemented with 200 μg/mlkanamycin sulfate. Fronds wre decontaminated from infecting Agrobacteriaby transferring the infected tissue to fresh medium with antibioticevery 2-4 days. The fronds were incubated on this medium forapproximately four weeks under conditions of low light (1-5 μM/m²·sec).

Duckweed nodule cultures for transformation were produced as follows.Duckweed fronds are separated, the roots are cut off with a sterilescalpel, and the fronds are placed, ventral side down, on Murashige andSkoog medium (catalog number M-5519; Sigma Chemical Corporation, St.Louis, Mo.) pH 5.6, supplemented with 5 μM 2,4-dichlorophenoxyaceticacid, 0.5 μM 1-Phenyl-3(1,2,3-thiadiazol-5-yl) urea thidiazuron (SigmaP6186), 3% sucrose, 0.4 Difco Bacto-agar (Fisher Scientific), and 0.15%Gelrite (Sigma). Fronds are grown for 5-6 weeks. At this time, thenodules (small, yellowish cell masses) appeared, generally from thecentral part of the ventral side. This nodule tissue was detached fromthe mother frond and cultured in Murashige and Skoog medium supplementedwith 3% sucrose, 0.4% Difco Bacto-agar, 0.15% Gelrite, 1 μM2,4-dichlorophenoxyacetic acid, and 2 μM benzyladenine.

Duckweed nodule cultures were transformed as follows. The appropriateAgrobacterium tumefaciens strain is grown on potato dextrose agar or YEBagar with 50 mg/L kanamycin and 100 μM acetosyringone, and resuspendedin Murashige and Skoog medium supplemented with 0.6 M Mannitol and 100μM acetosyringone. Nodule culture tissue was inoculated by immersing inthe solution of resuspended bacteria for 1-2 minutes, blotted to removeexcess fluid, and plated on co-cultivation medium consisting ofMurashige and Skoog medium supplemented with auxin and cytokininoptimized to promote nodule growth and 100 μM acetosyringone. See,Yamamoto et al. (2001) In Vitro Cell Dev. Biol. Plant 37:349-353.

For selection, nodule culture tissue was transferred to regenerationmedium Murashige and Skoog medium with 3% sucrose, 1 μM2,4-dichlorophenoxyacetate, 2 μM benzyladenine, 0.4% Difco Bacto-Agar,0.15% Gelrite 500 mg/L cefotaxime, and 200 mg/L kanamycin sulfate andcultured for approximately four weeks under continuous light (20-40μM/m² sec). The nodule tissue was transferred every 7 days to freshculture medium. Selection is complete when the nodule tissue showsvigorous growth on the selection agent. In some examples, thetransformed duckweed nodule cultures are transferred directly toregeneration medium for selection, instead of undergoing selection inco-cultivation medium.

For regeneration of transformed duckweed, the selected nodule culturewas transferred to regeneration medium (0.5× Schenk and Hildebrandtmedium supplemented with 1% sucrose and 200 mgs/L kanamycin) to organizeand produce plants. The nodule culture is incubated on regenerationmedium under full light for approximately 3 weeks, until fronds appear.Fully organized fronds were transferred to liquid Schenk and Hildebrandtmedium with 1-3% sucrose and incubated under full light for furtherclonal proliferation.

Detection of Biologically-Active Interferon Produced from DuckweedFronds or Duckweed Nodule Culture

Biologically-active interferon was detected by various assays, includinga solid phase sandwich immunoassay as described in Staehlin et al.(1981) Methods Enzymol. 79:589-594 and Kelder et al. (1986) MethodsEnzymol. 119:582-587, herein incorporated by reference, and a cytopathiceffect inhibition assay (described in Tovey et al. (1978) Nature276:270-272, herein incorporated by reference. Secreted interferon wascollected from the duckweed culture medium, while non-secretedinterferon was collected from ground-up or lysed duckweed plants orduckweed nodule tissue.

A solid phase sandwich immunoassay for interferon was performed using akit from PBL Laboratories (New Brunswick, N.J.) according to themanufacturer's instructions. Briefly, interferon is captured by anantibody bound to the microtiter plate wells. A second antibody is thenused to reveal the bound antibody. An anti-secondary antibody conjugatedto horseradish peroxidase (HRP) is then used to mark the interferon.Tetramethyl benzidine (TMB) initiates a peroxidase-catalyzed colorchange so that the interferon level can be observed and compared with astandard. A monoclonal antibody specific for α-2b-interferon (Cat. No.11105, PBL Laboratories) was used for this assay in the presentexamples.

A cytopathic effect inhibition assay was performed according to themethods of Tovey et al. (1978) Nature 276:270-272. Briefly, serialtwo-fold dilutions of the preparation to be assayed are diluted in a 96well microtiter plate (Falcon Inc) in a volume of 100 μl of Eaglesminimal essential medium (Life Technologies Inc) supplemented with 2%fetal calf serum (Life Technologies Inc) in parallel with serial twofold dilutions of the US National Institutes of Health human IFN alphainternational reference preparation (G-002-901-527). Twenty thousandhuman amnion cells (line WISH) are then added to each well of themicrotiter plate in a volume of 100 μl of medium with 2% fetal calfserum. The cells were incubated over-night in an atmosphere of 5% CO₂ inair at 37° C., the medium was removed and replaced with 200 μl of mediumwith 2% fetal calf serum containing vesicular stomatitis virus at amultiplicity of infection of 0.1. The cells were further incubatedover-night in an atmosphere of 5% CO₂ in air at 37° C. and thecytopathic effect due to virus replication was then evaluated under alight microscope. Interferon titers were expressed as the reciprocal ofthe IFN dilution which gave 50% protection against the cytopathiceffects of the virus. Interferon titers were expressed in internationalreference units by reference to the titer of the reference preparation.

The following examples demonstrate the expression of biologically activeinterferon variants in duckweed.

Example 1

A study was performed to determine culture IFN levels in media andtissue at various time points in a batch culture. A set of 20-30 ml 175oz.-culture jars were inoculated on Day 0 with 20 fronds of a linepreviously identified as expressing detectable levels of humanα-2b-interferon (IFN). The cultures were grown under autotrophic,buffered conditions with continuous high light provided byplant/aquarium fluorescent grow bulbs. At each time point—days 5, 7, 13,15, and 18—the fresh weight and media volume were measured for fourcultures. From each culture, media and tissue samples were obtained anda plant protease inhibitor cocktail was added. The tissue samples wereground and spun cold. The supernatant was collected. Media and tissueextracts were stored at −70° C. until all samples were collected. IFNlevels in media and tissue extracts were determined on the same dayusing the solid phase sandwich immunoassay described above. Totalculture IFN in media and tissue was calculated by multiplying measuredIFN concentrations and the volume of media and the fresh weight for theculture, respectively. FIG. 1 shows the relative IFN levels on days 7,13, 15, and 18 compared to day 5. The last time point represents theaverage value for three cultures instead of four due to loss of oneculture.

Example 2

A study was performed to determine culture IFN levels in media andtissue at various time points in a batch culture. A set of 24-30 ml 175oz.-culture jars were inoculated on Day 0 with 20 fronds of the sameline as in Example 1. The cultures were grown under autotrophic,unbuffered conditions with continuous high light provided byplant/aquarium fluorescent grow bulbs. At each time point—days 7, 10,12, 14, 17, and 19—the fresh weight and media volume were measured forfour cultures. From each culture, media and tissue samples were obtainedand a plant protease inhibitor cocktail was added. The tissue sampleswere ground and spun cold. The supernatant was collected. Media andtissue extracts were stored at −70° C. until all samples were collected.IFN levels in media and tissue extracts were determined on the same dayusing the solid phase sandwich immunoassay described above. Totalculture IFN in media and tissue was calculated by multiplying measuredIFN concentrations and the volume of media and the fresh weight for theculture, respectively. FIG. 2 shows the relative IFN levels on days 14,17, and 19 compared to day 12. Media IFN levels on day 7 and day 10 werebelow the range of the extended range protocol for the immunoassay.

Example 3

Duckweed lines transformed with the expression constructs listed inTable 2 were produced using the methods described above. Thesetransformed duckweed lines were grown for 14 days under autotrophicconditions. Bovine serum albumin at a concentration of 0.2 mg/ml wasincluded in the growth media. On day 14, media and tissue extracts wereprepared as described in Example 1, and the interferon levels in theseextracts were determined using the solid phase sandwich immunoassay asdescribed above. Table 3 gives the number of clonal duckweed linesassayed and the mean media interferon concentration for each expressionconstruct. Table 4 shows the interferon levels within the duckweedtissue for the duckweed lines transformed with the interferon expressionconstructs that did not contain a signal peptide (IFN01, IFN10, andIFN12). Both the mean interferon level for all clonal lines assayed forthe designated construct, and the interferon level for thetop-expressing line are shown.

TABLE 3 Mean Interferon Expression # of Clonal Lines ConcentrationConstruct Tested (ng/ml) IFN01 41 0 IFN02 75 2.3 IFN03 41 0.18 IFN05 442.5 IFN07 41 1.5 IFN08 41 1.4 IFN09 41 30.3 IFN10 41 0 IFN11 39 9.9IFN12 41 0

TABLE 4 Mean Value Top Expresser IFN % of total % of total Constructsoluble protein soluble protein IFN01 0.00003 0.0001 IFN10 0.000640.0074 IFN12 0.00014 0.001

The biological activity of the interferon produced by these transformedduckweed lines was assayed by the cytopathic effect inhibition assaydescribed above. Table 5 gives the results for the top expressing linefor each construct. The interferon activity is shown for the media forthose constructs containing a signal peptide and the tissue for thoseconstructs lacking a signal peptide.

TABLE 5 Top Expresser IFN Source IU/ml (media) Construct material IU/mgtotal protein (tissue) IFN01 Tissue 40 IFN02 Media 16,000 IFN03 Media320 IFN05 Media 6,400 IFN07 Media 6,000 IFN08 Media 3,200 IFN09 Media200,000 IFN10 Tissue 19,300 IFN11 Media 30,000 IFN12 Tissue 150

Example 4

A study was performed to determine the levels of IFN expressed fromtransgenic duckweed grown at bioproduction scales. Transgenic duckweedplants were generated using IFN expression constructs IFN02, IFN05,IFN09, IFN10, and IFN53 (see Table 2).

A minimum of 40 independent transgenic lines was screened for each ofthese constructs. The lines producing the highest levels of IFNexpression were analyzed further. The concentration of IFN in the mediaand tissue was determined by ELISA as described elsewhere herein.

Table 6 summarizes the IFN expression on both research and bioproductionscales for the constructs described above. Because Lemna is unique inthat it grows in a very dilute inorganic media with a low proteincontent, the expression values in this Table are defined by thepre-purification titer. In the case of IFN53, IFN represents over 30% ofthe total media proteins.

TABLE 6 Expression of IFN in Lemna Mean Average Media IFN concentrationfor Top Concentration for 2 Expressing Line (mg/L)^(c) Week ScreeningTrials Expres- Bio- (determined by ELISA)^(b) sion Research Researchpro- Tissue con- Scale Scale duction Media (mg/kg struct (2 weeks) (3weeks) Scale (mg/L)^(c) tissue)^(a) IFN02 2.0 — — 0.12 23.3 IFN05 1.1 —— 0.13 86.7 IFN09 24.8 60 30 1.51 164 IFN10 <0.1 — —  <0.0001 99.3 IFN53100 300 500 15.3^(d )  — ^(a)Based on 1 g of tissue yielding 20 mg ofprotein. ^(b)Based on recovery of 10 ml of media and 1 go of tissue perscreening trial. ^(c)Expressed as a pre-purification titer. ^(d)1 weekscreening trial.

The antiviral activity of the duckweed-produced IFN was determined asfollows. HuH7 cells were incubated for 24 hours at 37° C. with 1,000IU/ml of unpurified IFN from the duckweed media or Intron® A (Schering)as a control. The IFN was then removed and the cells were washed twice.The cells were subsequently infected with an RNA virus selected fromEncephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), orSindbis at a multiplicity of infection of 1.0 for 1 hour, at which timethe virus innoculum was removed. The cells were then washed three timesand allowed to grow for 24 hours at 37° C. The cells were harvested,lysed by six freeze/thaw cycles, and then cell debris was removed bycentrifugation. Serial dilutions of the virus were then assayed fortheir cytopathic effect on monkey CV1 Vero cells. The duckweed-producedIFN exhibited similar antiviral activity to that observed for Intron®A.

The antiproliferative activity of the duckweed-produced IFN wasdetermined as follows. Interferon-sensitive Daudi cells were seeded inmicrotiter plates at an initial concentration of 50,000 cells/ml. Theculture were then either left untreated or were treated with 1000 IU ofunpuri fled duckweed-produced IFN, Intron® A, or an equivalent volume ofcontrol media derived from non-transgenic plants grown under the sameconditions as for the transgenic plants. After four days, the number ofviable and dead cells were determined by the trypan blue-exclusionviability test.

Example 5

The sequence of the biologically active α-2b-interferon produced induckweed was determined by mass spectrometry. The duckweed-producedinterferon consisted of a mixture of five different species, withcarboxy-terminus truncations of 4-8 amino acids in comparison with wildtype human α-2b interferon. No full-length α-2b interferon was detected.The sequences of the variant interferons produced in duckweed are shownin SEQ ID NOS: 6-10. The predominant species produced was the 158 aminoacid polypeptide shown in SEQ ID NO:9. The sequences of thecorresponding precursor interferon polypeptides containing the humanα-2b-interferon signal peptide are shown in SEQ ID NOS:1-5. Although thepresent invention is not limited to any particular mechanism, it isbelieved that the interferon variants were produced in duckweed by theaction of an endogenous plant protease. It is the novel finding of thepresent invention that truncated interferon variants having the aminoacid sequences shown in SEQ ID NOS:6-10 are biologically active, asshown in the Examples above.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated polynucleotide encoding a polypeptide consisting of theamino acid sequence set forth in SEQ ID NO:10.
 2. An isolatedpolynucleotide encoding a polypeptide consisting of the amino acidsequence set forth in SEQ ID NO:5.
 3. An isolated polynucleotideencoding a polypeptide consisting of a signal peptide operably linked tothe amino acid sequence set forth in SEQ ID NO:10.
 4. An expressioncassette comprising the isolated polynucleotide of claim
 1. 5. A hostplant cell comprising the expression cassette of claim
 4. 6. The hostplant cell of claim 5, wherein said plant cell is a duckweed cell. 7.The isolated polynucleotide of claim 3, wherein said signal peptide is aplant signal peptide.
 8. An expression cassette comprising the isolatedpolynucleotide of claim
 3. 9. A host plant cell comprising theexpression cassette of claim
 8. 10. The host plant cell of claim 9,wherein said plant cell is a duckweed cell.